The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2000 BC. Until the late 1800s indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye's brilliant blue color lead to its development as one of the main articles of trade between Europe and the Far East. In 1883 Alfred von Baeyer identified the formula of indigo: C.sub.16 H.sub.10 N.sub.2 O.sub.2. In 1887 the first commercial chemical manufacturing process for indigo was developed. This process, still in use today, involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. The process' final product, indoxyl, oxidized spontaneously to indigo by exposure to air.
Current commercial chemical processes for manufacturing indigo result in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic by-products is desirable. One such method which results in less toxic by-product generation involves indigo biosynthesis by microorganisms.
Ensley et al. [(1983) Science 222:167-169] found that a DNA fragment from a transmissible plasmid isolated from the soil bacterium Pseudomonas putida enabled Escherichia coli stably transformed with a plasmid harboring the fragment to synthesize indigo in the presence of indole or tryptophan. Ensley et al. postulated that indole, added either as a media supplement or produced as a result of enzymatic tryptophan catabolism, was converted to cis-indole-2,3-dihydrodiol and indoxyl by the previously identified multi-component enzyme naphthalene dioxygenase (NDO) encoded by the P. putida DNA fragment. The indoxyl so produced was then oxidized to indigo upon exposure to air. The dioxygenase described by Ensley et al. is a preferred oxygenase useful in the production of indigo as further described herein.
NDO had previously been found to catalyze the oxidation of the aromatic hydrocarbon naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene [Ensley et al. (1982) J. Bacteriol. 149:948-954]. U.S. Pat. No. 4,520,103, incorporated by reference, describes the microbial production of indigo from indole by an aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprised of multiple components: a reductase polypeptide (Rd, molecular weight of approximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxin polypeptide (Fd, molecular weight of approximately 13 kD); and a terminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised of four subunits having an .alpha..sub.2.beta..sub.2 subunit structure (approximate subunit molecular weights: .alpha., 55 kD; .beta., 21 kD). ISP is known to bind naphthalene, and in the presence of NADH, Rd, Fd and oxygen, to oxidize it to cis-naphthalene-dihydrodiol. Fd is believed to be the rate-limiting polypeptide in this naphthalene oxidation catalysis, (see U.S. Pat. No. 5,173,425, incorporated herein by reference, for a thorough discussion of the various NDO subunits and ways to improve them for purposes of indigo biosynthesis).
In addition, aromatic dioxygenases other than NDO may also be useful in the biosynthetic production of indigo, for example, a toluene monooxygenase (TMO) such as that from Pseudomonas (P. mendocina) capable of degrading toluene was also able to produce indigo when the culture medium was supplemented with indole. For details, see U.S. Pat. No. 5,017,495, incorporated herein by reference. In principle, any enzyme capable of introducing a hydroxyl moiety into the 3-position of indole to give indoxyl is a candidate for use in the biosynthetic production of indigo.
It has also long been known that microorganisms contain biosynthetic pathways for the production of all 20 essential amino acids, including the aromatic amino acid L-tryptophan. The de novo synthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine) share a common pathway up through the formation of chorismic acid. After chorismic acid synthesis, specific pathways for each of the three aromatic amino acids are employed to complete their synthesis.
Bacterial biosynthesis of tryptophan from chorismic acid (specifically in E. coli) is under the control of the tryptophan (trp) operon. The (trp) operon, comprised of regulatory regions and several structural genes, has been extensively studied because of its complex and coordinated regulatory systems. The regulatory and structural organization of the E. coli trp operon, along with the catalytic activities encoded by the structural genes of the operon, appear in FIG. 1 of U.S. Pat. No. 5,374,543, incorporated herein by reference. U.S. Pat. No. 5,374,543 describes improvements in the intracellular production of indole, specifically as it relates to the conversion of indole-3'-glycerol-phosphate (InGP), in conjunction with L-serine, to L-tryptophan. The reaction is catalyzed by the multi-subunit enzyme tryptophan synthase. During the reaction, indole is produced as an intermediate. However, the indole is very rapidly combined with L-serine in a stoichiometric fashion to produce L-tryptophan. Thus, no free indole is produced as a result of this InGP plus L-serine conversion to tryptophan.
However, Yanofsky et al. [(1958) Biochim. Biophys. Acta. 28:640-641] identified a tryptophan synthase mutant which led to the accumulation of indole. This particular tryptophan synthase mutant, however, was subject to spontaneous reversion to the wild-type phenotype, as the mutation resulted from a single nucleotide base pair change in the gene coding for the .beta. subunit of tryptophan synthase.
U.S. Pat. No. 5,374,543 describes a method for creating stable tryptophan synthase mutants capable of yielding high levels of intracellular indole. When such indole accumulating tryptophan synthase mutants express an aromatic dioxygenase enzyme like NDO, the accumulated indole may be converted to indoxyl, which can then be oxidized to indigo by molecular oxygen. Thus, through the commercial application of recombinant DNA technology, by the overexpression of a modified trp operon capable of continuously producing indole and an oxygenase enzyme capable of simultaneous conversion of indole to indoxyl, indigo can be produced from a renewable raw material such as glucose.
In shake flask studies applicants have determined that during the synthesis of indigo from indole, low levels of other compounds or by-products accumulate in the culture supernatant. One of these by-products, isatin (indole 2,3-dione), has been found to inhibit the oxygenase (i.e., NDO) activity in the production strain and, consequently, reduces overall indigo production; thus, isatin is undesirable. In addition to the by-product isatin, indirubin, a red dye material derived from isatin, may be produced during this biosynthetic indigo production process. The by-product isatin is believed to reduce the productivity of the production strain, while the by-product indirubin is believed to cause undesirable dyeing characteristics to microbially produced indigo which is expressed as a red cast on cloth dyed with indirubin-tainted microbially produced indigo.
Because the production in shake flasks of one or more of these by-products may either reduce the productivity of this production strain and/or cause undesirable characteristics of the indigo produced therefrom, an object of the present invention is to reduce the buildup of isatin or remove such isatin formed as a by-product of biosynthesis of indigo in microbial cells. Removal of isatin will potentially enhance the overall production of indigo in a fermentor and reduce or prevent the accumulation of indirubin. One method to reduce the buildup of isatin or remove such isatin, as detailed herein, relates to the isolation, cloning, sequencing and expression in indigo-producing host strains of a gene encoding an enzyme having isatin-removing activity. Preferably the enzyme is an isatin hydrolase, an enzyme capable of degrading isatin; however, any method to remove or inhibit isatin formation is contemplated by the present invention. Thus, another aspect of the present invention is the enhanced production of biosynthetic indigo by reducing the buildup or removing accumulated isatin through means, including, but not limited to, enzymatic conversion of the isatin by contacting it with an isatin-removing enzyme such as an isatin hydrolase, general base catalyzed chemical conversion of the isatin at appropriate temperature and pH, or through adsorption of the isatin to carbon or a suitable resin. These aspects of the invention are detailed below.